Ca2+ release via IP3R channels is the most ubiquitous and versatile cellular signaling mechanism that plays a key role in the regulation of diverse physiological functions, including fertilization, hormone secretion, gene transcription, metabolic regulation, immune responses, apoptosis, learning and memory. Despite established significance of IP3Rs in physiology and pathology, the molecular mechanisms underlying function of these channels, both in native and disease states, remain poorly understood, mainly because of the lack of high- resolution structural details about the 3D architecture of IP3Rs. Such structures have proven exceptionally difficult to obtain given the large size of IP3Rs (~1.3 MDa), their location in the membrane environment, and their dynamic nature. The focus of this proposal is type 1 IP3R (IP3R1), the predominant type of IP3-gated Ca2+ release channel in cerebellar Purkinje cells. To date, the best structure of the entire IP3R1 is resolved by single-particle electron cryomicroscopy (cryo-EM) at intermediate resolution (10-15 ), and the crystal structures are limited to a soluble portion of the cytoplasmic region representing only ~15% of the overall structure. Therefore, most critical issues surrounding gating of IP3R channels are stil ambiguous. In this application, we seek to answer the fundamental questions on IP3R1 gating: what are the structural determinants of Ca2+ permeation through IP3R1, how ligands control the gating process and what conformational changes underlie pore opening in IP3R channels. To address these questions, we will extend structure determination of the entire IP3R1 to sub-nanometer resolution and will determine its structure in a near-native lipid environment. We will combine structural methodologies from cryo-EM, computational tools and bioinformatics with biochemical and biophysical techniques including radioligand binding, fluorescence- based Ca2+ flux assays and lipid bilayer channel recordings. This multidisciplinary approach will allow correlating structural analysis with channel function. Proposed structural studies will exploit single-particle cryo- EM methodology. Thus, the IP3R1 protein complex will be purified from detergent solubilized microsomal membranes and visualized in the form of individual particles embedded in vitreous ice. The IP3R1 structure will be analyzed in Apo- (aim 1) and ligand-bound states (aim 2) by reproducing the functionally relevant conditions in vitro and freeze-trapping the reaction on the EM grid. We then propose to reconstitute IP3R1 channel into small unilamellar lipid vesicles and to use a variant of single-particle reconstruction to solve the channel structure in the lipid membrane (aim 3). With these studies accomplished, we anticipate to establish the structural and mechanistic basis for IP3R1 function and to elucidate how defects in molecular mechanisms regulating the channel's gating can lead to abnormal cell Ca2+ levels underlying numerous diseases.